Hello, I'm Chinmay Lalgudi. I am a rising sophomore at Lynbrook High School in San Jose, California. I am really interested in biology, especially synthetic biology and research. I hope to develop my ideas and increase my knowledge throughout this camp. My interest in science and biology in particular really started to take shape in middle school, as my 7th grade science teacher sparked my interest in synthetic and cell biology, In the future, I would be interested in pursuing a career as researcher in evolutionary and synthetic biology, or as a a veterinarian. I also competed in Science Bowl in 7th and 8th grade, and won 1st place in the National Science Bowl in 2016. In addition to Science Bowl in middle and high school, I have competed in Science Olympiad 7th through 9th grades, and this year, I competed in the Ocean Science Bowl competition. Furthermore, I have science fair experience, competing in the Synopsys Science Fair for 4 years, and in the California State Science Fair in 9th grade. I love playing and watching soccer and basketball, and I also enjoy playing piano and violin.
The Overexpression of the EPO Gene to Regulate Red Blood Cell Production Regarding Extrinsic Hemolytic Anemia
By: Pranav Kakhandiki and Chinmay Lalgudi
Anemia is defined as the lack of healthy red blood cells or hemoglobin in the blood stream. There are several types of anemia, including aplastic anemia, iron deficiency anemia, vitamin deficiency anemia, sickle cell anemia, and hemolytic anemia. The type of anemia that our design project surrounds is hemolytic anemia. In hemolytic anemia, red blood cells are destroyed and removed from the bloodstream, before their normal lifespan ends.
Image result for hemolytic anemia
Red blood cells originate as immature cells in the bone marrow, before getting released into the bloodstream after 7 days of maturation. Red blood cells have a lifetime of 120 days in the bloodstream, before more RBC’s are produced by the bone marrow. However, in hemolytic anemia, the body is unable to produce enough RBC’s to replenish the past cells and sustain a healthy body. Common symptoms of hemolytic anemia include: dark urine, heart murmur, increased heart rate, and an enlarged liver. If left untreated, this disease is notorious to cause other severe problems in the body, like fatigue, arrhythmias, and heart failure.
There are two types of hemolytic anemia: extrinsic hemolytic anemia and intrinsic hemolytic anemia. Intrinsic hemolytic anemia is often inherited and familial, and develops when the RBC’s produced by the body are defective. Its effects may include issues with RBC proteins or oxidative stress handling. In the contrary, extrinsic hemolytic anemia is acquired and can be caused by a myriad of factors, including infections and cancers, but most often occurs when there is increased activity in the spleen, trapping and destroying a great quantity of RBC’s. Our project design targets extrinsic hemolytic anemia, because we wanted to find a way to produce more red blood cells, counteracting the effect the spleen has in the bloodstream.
When the bone marrow produces red blood cells, it uses a cytokine (signalling molecule) for erythrocyte precursors called erythropoietin. Erythropoietin is a glycoprotein and essentially controls erythropoiesis, regulating red blood cell production by promoting erythroid differentiation and initiating hemoglobin synthesis. This protein is found in plasma and is produced in interstitial fibroblasts in the kidney, while also being produced in perisinusoidal cells in the liver. The production of erythropoietin is more significant in the liver during the fetal and perinatal periods, while production in the kidney is much more prominent throughout adulthood.
Related image
The Epo Gene is a member of the EPO/TPO family and controls the erythropoietin production in the bloodstream. It is located on the long arm of Chromosome 7, in location 7q22.1.
As a reference, our design involves a series of steps: 1. Inserting the gene that produces erythropoietin in the kidney, and the promoter which allows it to function. 2. Inserting these genes inside a retrovirus, to be used as a viral vector. 3. Injecting a viral vector into the kidney, and allowing it to spread the genes in renal cells. 4. More erythropoietin is produced. 5. Erythropoietin travels through the bloodstream to the bone marrow. 6. More red blood cells are produced. 7. Overproduction of RBC’s from bone marrow and increased destruction of RBC’s by spleen equal a normal amount of RBC’s.
To go into more detail, the EPO promoter will be placed in front of the actual gene that codes for producing erythropoietin. Our design will contain multiple copies of the original gene found in the kidney, along with synthetic promoters (inside the retrovirus). Repeating the gene multiple times causes the RNA polymerase to transcribe it many times, creating more of the targeted proteins, which in this case, is erythropoietin. This will effectively overexpress the gene as shown in the diagram. In the diagram, the copies of the EPO gene are being inserted by a viral vector (retrovirus). The EPO promoters are added in addition, allowing for the transcription of the EPO gene. After excess erythropoietin is produced in the kidney, it travels through the bloodstream, and into the bone marrow. In the bone marrow, erythropoietin acts as a cytokine for erythrocyte precursors and signals for the production of red blood cells, leading to an excess in the production of red blood cells. As mentioned before, in extrinsic hemolytic anemia, however, the spleen traps and destroys large masses of red blood cells, so the excess of these will result in a normal and healthy amount of red blood cells.
Increased Erythropoietin
Increased RBC production
Normal amount of RBC’s
0
0
0
1
1
1
While this treatment is practically permanent, monitoring the progress of this gene therapy is essential. There are 2 main ways to track the development of this procedure: Red Blood Cell Counter and Mean Corpuscular Volume. Both of these techniques are widely used to diagnose anemia, and now we plan on using them as a verification that the treatment is indeed working. A Red Blood Cell Counter usually monitors how many RBC’s there are in a certain blood sample, so they will be used to monitor that RBC’s are really being overproduced in our treatment. MCV (Mean Corpuscular Volume) tests for the average volume of RBC’s. These can be directly measured by automated hematology analyzer, and will be used in our project again to validate this new treatment.
Image result for red blood cell counter machine
When being diagnosed with Extrinsic Hemolytic Anemia, there are existing treatments at medical facilities. Each treatment can help patients with different needs, as they have their advantages and disadvantages in their own way. A blood transfusion is the process of receiving blood into one's circulation intravenously. Although blood transfusions can significantly decrease the blood loss, they have an elevated amount of contamination and disease transmission cases between patients. Plasmapheresis is another procedure used to “filter” the blood from the body. Plasma is extracted from blood cells, treated or replaced, and returned back into the bloodstream. Plasmapheresis has little side effects, but constant trips to medical facilities are needed to replace the plasma from the bloodstream. The most common treatment is medicine, but its use has declined due to its severe unwanted side effects and the potential disability to make antibodies against red blood cells. Surgical procedures to treat hemolytic anemia are expensive and require many follow-up trips, while also posing the risk to complicate issues further in the body and later on in life. The newest treatment for this fatal disease is Bone Marrow/Stem Cell Therapy. This therapy has a lot of potential for the future, and researchers are still trying to understand fully how it works. This new technology also come at a disadvantage, because of the unknown long-term side effects and the high chance of rejection from the body.
Image result for plasmapheresis
2017-07-19 (3).png
Like any other therapy, the treatment designed by us has its profits and drawbacks too. Some of the edges it has on the other technologies include the permanent nature of this treatment (injection into kidney) and today’s lowering cost of gene therapy. But, it also has some potential problems. Firstly, with the overproduction of RBC’s, the spleen could end up killing even more RBC’s to balance the high concentrations of erythropoietin. More importantly, the bone marrow may not be able to support the amount of erythropoietin produced, resulting again in a lack of RBC’s in the bloodstream.
In conclusion, hemolytic anemia is a major problem all around the world, and it is our responsibility to treat it. Therapies today have lackluster results, while possessing severe and unknown side effects. The treatment devised by us increases the production of erythropoietin in the kidney, directly influencing the production of red blood cells in the bone marrow. Although this technology does have its setbacks, this treatment of using gene therapy utilizing viral vectors to treat Hemolytic Anemia could revolutionize the world and save millions of lives.
Bibliography
Administrator. Gene Therapy Retrovirus Vectors Explained. N.p., n.d. Web. 21 July 2017.
"Anemia." Mayo Clinic. Mayo Foundation for Medical Education and Research, 06 Aug. 2016. Web. 21 July 2017.
Database, GeneCards Human Gene. "EPO Gene(Protein Coding)." GeneCards Is a Searchable, Integrative Database That Provides Comprehensive, User-friendly Information on All Annotated and Predicted Human Genes. N.p., n.d. Web. 21 July 2017.
Database, GeneCards Human Gene. "EPO Gene(Protein Coding)." GeneCards Is a Searchable, Integrative Database That Provides Comprehensive, User-friendly Information on All Annotated and Predicted Human Genes. N.p., n.d. Web. 21 July 2017.
Ebert, Benjamin L., and H. Franklin Bunn. "Regulation of the Erythropoietin Gene." Blood. American Society of Hematology, 15 Sept. 1999. Web. 21 July 2017.
"Expression Vector." Wikipedia. Wikimedia Foundation, 08 July 2017. Web. 21 July 2017.
"Hemolytic Anemia." Hemolytic Anemia | Johns Hopkins Medicine Health Library. N.p., n.d. Web. 21 July 2017.
"Hemolytic Anemia." Wikipedia. Wikimedia Foundation, 17 July 2017. Web. 21 July 2017.
Jelkmann, Wolfgang. "Regulation of Erythropoietin Production." The Journal of Physiology. Blackwell Science Inc, 15 Mar. 2011. Web. 21 July 2017.
Kahn, April, and Rachel Nall. "Hemolytic Anemia." Healthline. Healthline Media, 23 Oct. 2015. Web. 21 July 2017.
"The LightSwitch Promoter Reporter GoClone Collection." The LightSwitch Promoter Reporter GoClone Collection | Active Motif LightSwitch Store. N.p., n.d. Web. 21 July 2017.
MPH, Siamak N. Nabili MD. "What Is Erythropoietin (EPO)? Test, Definition, Side Effects." MedicineNet. N.p., n.d. Web. 21 July 2017.
Robbins, P. D., and S. C. Ghivizzani. "Viral Vectors for Gene Therapy." Pharmacology & Therapeutics. U.S. National Library of Medicine, Oct. 1998. Web. 21 July 2017.
"Types of Hemolytic Anemia." National Heart Lung and Blood Institute. U.S. Department of Health and Human Services, 21 Mar. 2014. Web. 21 July 2017.
The Overexpression of the EPO Gene to Regulate Red Blood Cell Production Regarding Extrinsic Hemolytic Anemia
By: Pranav Kakhandiki and Chinmay Lalgudi
Anemia is defined as the lack of healthy red blood cells or hemoglobin in the blood stream. There are several types of anemia, including aplastic anemia, iron deficiency anemia, vitamin deficiency anemia, sickle cell anemia, and hemolytic anemia. The type of anemia that our design project surrounds is hemolytic anemia. In hemolytic anemia, red blood cells are destroyed and removed from the bloodstream, before their normal lifespan ends.
Red blood cells originate as immature cells in the bone marrow, before getting released into the bloodstream after 7 days of maturation. Red blood cells have a lifetime of 120 days in the bloodstream, before more RBC’s are produced by the bone marrow. However, in hemolytic anemia, the body is unable to produce enough RBC’s to replenish the past cells and sustain a healthy body. Common symptoms of hemolytic anemia include: dark urine, heart murmur, increased heart rate, and an enlarged liver. If left untreated, this disease is notorious to cause other severe problems in the body, like fatigue, arrhythmias, and heart failure.
There are two types of hemolytic anemia: extrinsic hemolytic anemia and intrinsic hemolytic anemia. Intrinsic hemolytic anemia is often inherited and familial, and develops when the RBC’s produced by the body are defective. Its effects may include issues with RBC proteins or oxidative stress handling. In the contrary, extrinsic hemolytic anemia is acquired and can be caused by a myriad of factors, including infections and cancers, but most often occurs when there is increased activity in the spleen, trapping and destroying a great quantity of RBC’s. Our project design targets extrinsic hemolytic anemia, because we wanted to find a way to produce more red blood cells, counteracting the effect the spleen has in the bloodstream.
When the bone marrow produces red blood cells, it uses a cytokine (signalling molecule) for erythrocyte precursors called erythropoietin. Erythropoietin is a glycoprotein and essentially controls erythropoiesis, regulating red blood cell production by promoting erythroid differentiation and initiating hemoglobin synthesis. This protein is found in plasma and is produced in interstitial fibroblasts in the kidney, while also being produced in perisinusoidal cells in the liver. The production of erythropoietin is more significant in the liver during the fetal and perinatal periods, while production in the kidney is much more prominent throughout adulthood.
The Epo Gene is a member of the EPO/TPO family and controls the erythropoietin production in the bloodstream. It is located on the long arm of Chromosome 7, in location 7q22.1.
As a reference, our design involves a series of steps: 1. Inserting the gene that produces erythropoietin in the kidney, and the promoter which allows it to function. 2. Inserting these genes inside a retrovirus, to be used as a viral vector. 3. Injecting a viral vector into the kidney, and allowing it to spread the genes in renal cells. 4. More erythropoietin is produced. 5. Erythropoietin travels through the bloodstream to the bone marrow. 6. More red blood cells are produced. 7. Overproduction of RBC’s from bone marrow and increased destruction of RBC’s by spleen equal a normal amount of RBC’s.
To go into more detail, the EPO promoter will be placed in front of the actual gene that codes for producing erythropoietin. Our design will contain multiple copies of the original gene found in the kidney, along with synthetic promoters (inside the retrovirus). Repeating the gene multiple times causes the RNA polymerase to transcribe it many times, creating more of the targeted proteins, which in this case, is erythropoietin. This will effectively overexpress the gene as shown in the diagram. In the diagram, the copies of the EPO gene are being inserted by a viral vector (retrovirus). The EPO promoters are added in addition, allowing for the transcription of the EPO gene. After excess erythropoietin is produced in the kidney, it travels through the bloodstream, and into the bone marrow. In the bone marrow, erythropoietin acts as a cytokine for erythrocyte precursors and signals for the production of red blood cells, leading to an excess in the production of red blood cells. As mentioned before, in extrinsic hemolytic anemia, however, the spleen traps and destroys large masses of red blood cells, so the excess of these will result in a normal and healthy amount of red blood cells.
While this treatment is practically permanent, monitoring the progress of this gene therapy is essential. There are 2 main ways to track the development of this procedure: Red Blood Cell Counter and Mean Corpuscular Volume. Both of these techniques are widely used to diagnose anemia, and now we plan on using them as a verification that the treatment is indeed working. A Red Blood Cell Counter usually monitors how many RBC’s there are in a certain blood sample, so they will be used to monitor that RBC’s are really being overproduced in our treatment. MCV (Mean Corpuscular Volume) tests for the average volume of RBC’s. These can be directly measured by automated hematology analyzer, and will be used in our project again to validate this new treatment.
When being diagnosed with Extrinsic Hemolytic Anemia, there are existing treatments at medical facilities. Each treatment can help patients with different needs, as they have their advantages and disadvantages in their own way. A blood transfusion is the process of receiving blood into one's circulation intravenously. Although blood transfusions can significantly decrease the blood loss, they have an elevated amount of contamination and disease transmission cases between patients. Plasmapheresis is another procedure used to “filter” the blood from the body. Plasma is extracted from blood cells, treated or replaced, and returned back into the bloodstream. Plasmapheresis has little side effects, but constant trips to medical facilities are needed to replace the plasma from the bloodstream. The most common treatment is medicine, but its use has declined due to its severe unwanted side effects and the potential disability to make antibodies against red blood cells. Surgical procedures to treat hemolytic anemia are expensive and require many follow-up trips, while also posing the risk to complicate issues further in the body and later on in life. The newest treatment for this fatal disease is Bone Marrow/Stem Cell Therapy. This therapy has a lot of potential for the future, and researchers are still trying to understand fully how it works. This new technology also come at a disadvantage, because of the unknown long-term side effects and the high chance of rejection from the body.
Like any other therapy, the treatment designed by us has its profits and drawbacks too. Some of the edges it has on the other technologies include the permanent nature of this treatment (injection into kidney) and today’s lowering cost of gene therapy. But, it also has some potential problems. Firstly, with the overproduction of RBC’s, the spleen could end up killing even more RBC’s to balance the high concentrations of erythropoietin. More importantly, the bone marrow may not be able to support the amount of erythropoietin produced, resulting again in a lack of RBC’s in the bloodstream.
In conclusion, hemolytic anemia is a major problem all around the world, and it is our responsibility to treat it. Therapies today have lackluster results, while possessing severe and unknown side effects. The treatment devised by us increases the production of erythropoietin in the kidney, directly influencing the production of red blood cells in the bone marrow. Although this technology does have its setbacks, this treatment of using gene therapy utilizing viral vectors to treat Hemolytic Anemia could revolutionize the world and save millions of lives.
Bibliography
Administrator. Gene Therapy Retrovirus Vectors Explained. N.p., n.d. Web. 21 July 2017.
"Anemia." Mayo Clinic. Mayo Foundation for Medical Education and Research, 06 Aug. 2016. Web. 21 July 2017.
Database, GeneCards Human Gene. "EPO Gene(Protein Coding)." GeneCards Is a Searchable, Integrative Database That Provides Comprehensive, User-friendly Information on All Annotated and Predicted Human Genes. N.p., n.d. Web. 21 July 2017.
Database, GeneCards Human Gene. "EPO Gene(Protein Coding)." GeneCards Is a Searchable, Integrative Database That Provides Comprehensive, User-friendly Information on All Annotated and Predicted Human Genes. N.p., n.d. Web. 21 July 2017.
Ebert, Benjamin L., and H. Franklin Bunn. "Regulation of the Erythropoietin Gene." Blood. American Society of Hematology, 15 Sept. 1999. Web. 21 July 2017.
"Expression Vector." Wikipedia. Wikimedia Foundation, 08 July 2017. Web. 21 July 2017.
"Hemolytic Anemia." Hemolytic Anemia | Johns Hopkins Medicine Health Library. N.p., n.d. Web. 21 July 2017.
"Hemolytic Anemia." Wikipedia. Wikimedia Foundation, 17 July 2017. Web. 21 July 2017.
Jelkmann, Wolfgang. "Regulation of Erythropoietin Production." The Journal of Physiology. Blackwell Science Inc, 15 Mar. 2011. Web. 21 July 2017.
Kahn, April, and Rachel Nall. "Hemolytic Anemia." Healthline. Healthline Media, 23 Oct. 2015. Web. 21 July 2017.
"The LightSwitch Promoter Reporter GoClone Collection." The LightSwitch Promoter Reporter GoClone Collection | Active Motif LightSwitch Store. N.p., n.d. Web. 21 July 2017.
MPH, Siamak N. Nabili MD. "What Is Erythropoietin (EPO)? Test, Definition, Side Effects." MedicineNet. N.p., n.d. Web. 21 July 2017.
Robbins, P. D., and S. C. Ghivizzani. "Viral Vectors for Gene Therapy." Pharmacology & Therapeutics. U.S. National Library of Medicine, Oct. 1998. Web. 21 July 2017.
"Types of Hemolytic Anemia." National Heart Lung and Blood Institute. U.S. Department of Health and Human Services, 21 Mar. 2014. Web. 21 July 2017.
Presentation Link: https://docs.google.com/presentation/d/1qLxqsEZv1Bv_s3Iiu-BrS1qBUi_KqEEvNlenOEU342E/edit#slide=id.g249ce713a4_2_98